Exosome for stimulating t cell and pharmaceutical use thereof

ABSTRACT

The present invention relates to an exosome for stimulating T cells and the pharmaceutical use thereof. Immune exosomes secreted from artificial antigen-presenting cells which express HLA, CD32, and co-stimulatory molecules CD32, CD80, CD83, and 4-1BBL are used to stimulate naive CD8+ T cells whereby preventive and therapeutic effects on tumors, pathogen infections, or autoimmune diseases can be provided.

BACKGROUND 1. Field of the Invention

The present invention relates to an exosome for stimulating T cells,which expresses HLA and co-stimulatory molecules, and the pharmaceuticaluse thereof.

2. Discussion of Related Art

Naive antigen-specific T cells may be directly stimulated byantigen-pulsed mature dendritic cells (DCs). Further, dendritic cellsrelease antigen-presenting vesicles, so-called “exosomes” by processingantigens in endosomal compartments capable of binding to a cellmembrane, e.g. multivesicular endosomes. A dendritic cell-derivedexosome (DEX) is an effective stimulant of T cells. A more effectivemeans of T cell activation by DEX appears to occur indirectly followingDEX interactions with DCs. Two major mechanisms have been proposed toexplain whether the antigen peptide-MHC-containing DEX may induceindirect antigen presentation in T cells by antigen presenting cells(APCs). First, by a process known as “cross-dressing”, the DEX bindsdirectly to the surface membrane of a receptor APC, transfers thepeptide-MHC complex thereof to the APC membrane, and then is recognizedby T cells without additional antigen processing. Second, the indirectpresentation mechanism takes place by transferring the antigen to APCMHCs in the DEX MHC via the capture and re-processing of the DEXpeptide-MHC complex by the APC. However, the generation of sufficientDEXs from the autologous DCs remains a barrier to the wide use of theDEX for immunotherapy.

The development of latex beads capable of being coupled toco-stimulatory molecules heralded a new area in artificialantigen-presenting cell (AAPC) technology. This approach enables precisecontrol of signaling, but has some limitations. In particular, theinteraction between T cells and beads is different from the interactionbetween T cells and natural APCs. The concept that a modified lipidsurface may improve immune synapse formation is attractive, andpreparations such as AAPCs began to be evaluated in recent studies. Anadvantage of cellular AAPCs is that once the cellular AAPCs are created,the cell line thereof may be approved and deposited, and thus isprovided as a source of a preparation that may be easily accessed for along period of time in order to be used for the production or expansionof T cells without any preparation of feeder cells, which is frequentlyrequired for methods of culturing autologous APCs or other T cells. Inparticular, mesenchymal stem cells, neural stem cells, embryonic stemcells, umbilical cord blood-derived cells, and chronic myelogenousleukemia K562 cell lines have been used for this purpose because they donot express endogenous HLA class I and II molecules. The presentinvention facilitates a detailed analysis of contribution of thesemolecules to the proliferation of T cells by introducing variousco-stimulatory molecules into K562 cells in order to additionallyenhance signalling. For example, after CD8+ T cells are stimulated byK562 cells into which HLA-A2, CD32, CD80, and CD83 are introduced, CD8+T cells specific for HLA-A2-restricted epitopes may be produced fromMART1. In order to produce antigen-specific T cells for adoptiveimmunotherapy, several gene modified K562-based AAPCs have been used.K562 cells have been widely used as a high releasing model for anexosome which expresses a general exosome marker such as CD9, CD63,CD81, CD82, and Tsg101. Furthermore, an immunosome serving as avirus-like particle for non-specifically stimulating T cells in vitrofrom HEK293 cells by variation of T cell co-stimulatory ligands having aglycosylphosphatidylinositol (GPI) anchor that promotes the localizationof a single-chain Fv (scFv) fragment of an OKT3 antibody and a lipidraft was developed.

The inventors established a hypothesis that K562 cells transduced with avector which expresses not only CD32, CD80, CD83, and 4-1BBL, but alsoHLA-A2 would naturally release immunological exosomes comparable to DEX,and characterized the phenotype of an exosome derived from modified K562cells, thereby completing the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is directed to providing an exosomeexhibiting an immunostimulatory reaction isolated from artificialantigen presenting cells which express HLA and co-stimulatory molecules,and the pharmaceutical use thereof.

To achieve the object, the present invention provides an exosome whichexpresses a human leukocyte antigen (HLA), CD32, CD80, CD83, and 4-1BBL.

The present invention also provides an immunotherapeutic agent includingthe exosome.

The present invention also provides a vaccine for preventing tumors,pathogen infections, or autoimmune diseases, including the exosome.

The present invention also provides a pharmaceutical composition fortreating tumors, pathogen infections, or autoimmune diseases, includingthe exosome.

The present invention also provides a method for proliferating T cells,the method including a step of co-culturing the exosome and any one Tcell of a CD4 T cell, a CD8 T cell, or a γδT cell.

The present invention also provides a method for preparing cytotoxic Tcells in vitro, the method including a step of stimulating any one of aCD4 T cell, a CD8 T cell, or a γδT cell with the exosome sensitized withone or more antigens selected from the group consisting of a tumorantigen, a pathogen antigen, and an autoantibody.

The present invention uses immunological exosomes secreted fromartificial antigen-presenting cells which express HLA and co-stimulatorymolecules to stimulate naive CD8+ T cells whereby preventive andtherapeutic effects on tumors, pathogen infections, or autoimmunediseases can be provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a result of characterization of a K562-derivedexosome (CoEX-A2) which expresses HLA-A2 and co-stimulatory molecules,and in FIG. 1a , K562 cells were transduced with a lentiviral vectorcarrying genes encoding HLA-A2, CD32, CD80, CD83, and CD137L(4-1BBL) andcultured without any antibiotic for 2 months by sorting the cells with aMoFlo cytometer to isolate single-cell clones, protein expression wasdetected by flow cytometry, and the histograms show protein expressionlevels in an A2-CD32-80-83-41BBL cell line (black) and aCD32-80-83-41BBL cell line (charcoal grey) as compared to K562 mothercells (bright grey). FIG. 1b illustrates the results of culturing CD8+ Tcells isolated from healthy volunteers together with anA2-CD32-80-83-41BBL (CoAPC-A2) cell line, a CD32-80-83-41BBL (CoAPC)cell line, and K562 cells and analyzing the cells in the presence (+) orabsence (−) of a CMV peptide. Additionally, the cells were analyzed byusing ModFit LT 3.0 software (Verity Software House). Representativestaining from three independent experiments are shown. CD8+ T cells(5×10⁵) isolated from human peripheral blood mononuclear cells (PBMCs)were activated by using artificial antigen-presenting cells. FIG. 1cillustrates flow cytometry analysis results of the expression ofHLA-A2/co-stimulatory molecules of CoEX-A2 or the K562 cell-derivedexosome (KEX). FIG. 1d illustrates flow cytometry analysis results ofexosomes coupled to latex beads coated with diverse exosome-specificmarker antibodies, and for comparison, latex beads coated with an entireexosome preparation are included. The plot is a representation of theintensities derived from a control (grey) with the only correspondingbead and an exosome-specific antibody (black). Each plot is arepresentative value from three independent experiments. FIG. 1eillustrates dot blot analysis results of CoEX-A2 lysates, thecorresponding dot was evaluated by using an exosome antibody array kit,and the exosome-specific antibody spots provided signals at variousdegrees. The value is an average of three independent experimentsperformed in triplicate, and the error bar is SEM.

FIG. 2 illustrates the efficient activation and proliferation of CD8+ Tcells by using a K562-derived exosome (CoEX) which has no HLA-A2 andexpresses CD32 and co-stimulatory molecules, FIG. 2a illustrates cellproliferation results measured after mixing CoEX or KEX with CD8+ Tcells at a ratio of 1:2 in the presence and absence of an anti-CD3antibody (hOKT3, 0.5 μg/mL) and stimulating the mixture with OKT3 andIL-2 for 16 hours, and FIG. 2b illustrates flow cytometry histogramsillustrating results compared with DYNBEAD including CD3 and aco-stimulatory molecule CD28, and additionally illustrates the intensityof CF SE-labeled cells analyzed by ModFit LT 3.0 software as arepresentative staining result in three independent experiments.

FIG. 3 illustrates a procedural view (left view) of stimulating γδTcells for 14 days, a result of proliferating γδT cells (right view), anda flow cytometry analysis result of Vγ9+/Vδ2+T (bottom view) by treatinghuman peripheral blood with zoledronic acid and IL-2 (1000 IU/mL) for 7days to increase the number of γδT cells, and then using CoEX or feedercells CoAPC and OKT3 (0.5 μg/mL) from Day 7.

FIG. 4 illustrates the results of confirming immunostimulatory responsesof a K562-derived exosome (CoEX-A2) which expresses HLA-A2 andco-stimulatory molecules, and FIG. 4a illustrates that CD8+ T cells aremeasured by flow cytometry after the CD8+ T cells are incubated with anexosome, analyzed in the presence (+) or absence (−) of a CMV peptide,and incubated with exosomes including HLA-A2/co-stimulatory molecules orincluding no HLA-A2/co-stimulatory molecules for 130 hours, andadditionally illustrates the intensity of CFSE-labeled cells analyzed byModFit LT 3.0 software as a representative staining result in threeindependent experiments. FIG. 4b illustrates ELISPOT analysis results ofstimulating CD8+ T cells (5×10⁵) isolated from human peripheral bloodmononuclear cells (PBMCs) by using exosomes (CoEX-A2, DEX) andantigen-presenting cells (CoAPC-A2, dendritic cell) in the presence orabsence of a pp65 peptide.

FIG. 5 illustrates ELISPOT analysis results showing the stimulation ofnaive CD8+ T cells by using a peptide-loaded exosome (CoEX-A2) whichexpresses HLA-A2 and co-stimulatory molecules. An exosome pre-loadedwith a viral peptide induces the dose-dependent stimulation of CD8+ Tcells. The K562 cell which expresses HLA-A2 and co-stimulatory moleculeswas used as a positive control.

FIG. 6 illustrates an inter-donor comparison of IFN-γ secretion andexpression by human CD8+ T cells treated with virus or tumorantigen-loaded exosomes. FIG. 6a shows a comparison of activities of CMVantigen-specific CD8+ T cells treated with an exosome (CoEX-A2) whichexpresses pp65₄₉₅₋₅₀₃-loaded HLA-A2 and co-stimulatory molecules for thestimulation of CD8+ antigen-specific T cells from five HLA-A2+ and 2HLA-A2-volunteers. FIG. 6b illustrates results in which CD8+ T cells areincubated with CoEX-A2 in the presence (+) or absence (−) of aMART1₂₆₋₃₅ peptide.

FIG. 7 shows results of evaluating indirect effects of an exosome byexamining the transfer of exosome surface molecules to CD8+ T cells orK562 cells, and FIG. 7a is a view illustrating the uptake of K562cell-derived exosomes modified by non-specific CD8+ T cells causing thestimulation of antigen-specific CD8+ T cell responses. CD8+ T cellstreated with an exosome which expresses HLA-2 and a co-stimulatorymolecule (CoEX-A2) obtained HLA-A2 and the co-stimulatory molecule ofthe exosome to cause the stimulation of an antigen-specific CD8+ T cellresponse by HLA-A2/TCR (Signal I), CD83, 41BBL, and CD80 co-stimulation(Signal II). FIG. 7b is a view illustrating that the uptake of 4 typesof exosomes by K562 cells enables K562-mediated induction ofantigen-specific CD8+T lymphocyte activation.

FIG. 8b illustrates the proliferation results of CD8+ cells stimulatedby an exosome (CoEX-A2) which expresses pp65 or MART1 peptide-pulsedco-stimulatory molecules and HLA-A2 molecules, FIG. 8b illustrates theIFN-γ ELISPOT analysis results performed by using pp65 or MART1peptide-pulsed CoEX-A2 in order to measure the frequency ofantigen-specific T cells, and FIG. 8c illustrates the flow cytometryanalysis results thereof, and CD8+ T cells were stimulated by CD80,CD83, and the 4-1BB ligand (4-1BBL; known as CD137L) for theproliferation of clones. In vitro expansion was achieved by the additionof the modified exosome which expresses feeder cells or co-stimulatoryligands.

FIG. 9 is a set of results illustrating that exosomes (CoEX-A2/pp65)which express HLA-A2 and co-stimulatory molecules and are loaded withpp65 exhibit CD8+ T cell stimulation at a level similar to that ofdendritic cell-derived exosome (DEX)/pp65, and FIG. 9a illustrates FITCvs. SSC dot plot results for CFSE-labeled T2 cells (target cells). Thegraph exhibits the spontaneous apoptotic rate in a normal sample at anE:T ratio of 200:1 and represents CD8+ T cell activity. When T2 cellswere cultured in the absence of a pp65 peptide, there was littleapoptosis (less than 1.8%). When the ratio of antigen-specific CD8+ Tcells:T2 cells was 200:1, the T cell apoptotic rate was increased upto >38%. FIG. 9b illustrates the degree of CTL toxicity according to theratio of antigen-specific CD8+ T cells:T2 cells (that is, effector cellvs. target cell). T2 cells were labeled with CFSE and co-cultured withCTL at the ratio and at 37° C. for 6 hours. At the end of theexperiment, dead cells were labeled with 7-AAD. The percentage of T cellapoptosis was measured by using flow cytometry. There was no significantdifference in cell apoptosis between cells treated with CoEX-A2 and DEX.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, the configuration of the present invention will bedescribed in detail.

The present invention relates to exosomes which express a humanleukocyte antigen (HLA), CD32, CD80, CD83, and 4-1BBL.

The present invention is characterized by providing exosomes asextracellular vesicles which are secreted from artificialantigen-presenting cells constructed so as to induce an antigen-specificcytotoxic T cell response and express HLA and co-stimulatory moleculesCD32, CD80, CD83, and 4-BBL while performing the functions of autologousantigen-presenting cells as an alternative to solve the disadvantages inthat conventional antigen-presenting cells, for example, dendritic cellsare present in small amounts in human peripheral blood mononuclear cellsand it is difficult to obtain a large amount of cells for clinicalapplication.

As used herein, the term “artificial antigen-presenting cells (aAPCs)”refers to antigen-presenting cells artificially constructed, and thecells are non-immune cells modified so as to express immune molecules.The aAPC which expresses MHC Class I or II (MHC I or II) either alone ortogether with other accessory molecules (co-stimulatory molecules and/oradhesion molecules) is used to study various aspects of T cell activatedcells which may be easily cultured in vivo, such as a cell line of tumorcells or fibroblasts. For the purpose of the present invention, theabove term refers to cells which do not express HLA Class I and IImolecules, for example, K562 cells, or cells in which nucleic acidsencoding HLA and co-stimulatory molecules CD32, CD80, CD83, and 4-1BBLare injected into recombinant 293T cells genetically engineered so asnot to express HLA Class I and II molecules, but is not limited thereto.

The term “co-stimulatory molecule” refers to a substance participatingin the interaction between receptor-ligand pairs and T cells, which areexpressed on the surface of antigen-presenting cells, and in order toinduce the expression and proliferation of cytokine genes, two or moresignals are required for resting T cells, the first signal is a signalimparting specificity and is produced by the interaction between anMHC/peptide complex and a TCR/CD3 complex, and the second signal isnon-specific to antigen and refers to a “co-stimulatory” signal. Thesignal is known as an activity provided by bone-marrow-derived accessorycells such as macrophages and dendritic cells. The co-stimulatorymolecules perform complete activation of CD8+ T cells by mediatingco-stimulatory signals required under normal physiological conditions.In the present invention, a combination of CD32, CD80, CD83, and 4-1BBLis used as the co-stimulatory molecule.

The exosomes are secreted from artificial antigen-presenting cellsprepared by selecting and introducing nucleic acids which encode HLA;and co-stimulatory molecules CD32, CD80, CD83, and 4-1BBL into cellswhich do not express HLA Class I and II molecules by using a knowntransformation technology, and thus may be isolated by centrifuging acell culture broth. According to one embodiment, artificialantigen-presenting cells may be prepared by using K562 cells as thecells which do not express HLA Class I and II molecules and into which avector into which nucleic acids encoding HLA and co-stimulatorymolecules are inserted is introduced.

The nucleic acids encoding HLA and the co-stimulatory molecules are usedin the broadest sense, and encompass single-stranded (ss) DNA,double-stranded (ds) DNA, cDNA, (−)-RNA, (+)-RNA, dsRNA, and the like.Preferably, the nucleic acid is double-stranded DNA.

Preferably, the HLA may be a human-derived nucleic acid sequence. Forexample, the HLA may be a base sequence set forth in SEQ ID NO: 1, butis not particularly limited thereto.

The CD80 may be a human- or mouse-derived nucleic acid sequence. Forexample, the CD80 may be a base sequence set forth in SEQ ID NO: 2, butis not particularly limited thereto.

The CD83 may be a human- or mouse-derived nucleic acid sequence, and maybe, for example, a base sequence set forth in SEQ ID NO: 3, but is notparticularly limited thereto.

The 4-1BBL may be a human- or mouse-derived nucleic acid sequence, andmay be, for example, a base sequence set forth in SEQ ID NO: 4, but isnot particularly limited thereto.

When a DNA is selected as the nucleic acid encoding HLA or theco-stimulatory molecule, the DNA may be used in a form in which the DNAis inserted into an expression vector.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (for example, bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (for example,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operably linked. Asused herein, the vector refers to a “recombinant expression vector” (orsimply, “expression vector”). In general, expression vectors useefulinrecombinant DNA techniques are predominantly in the form of plasmids,and “plasmid” and “vector” may be used interchangeably as the plasmid isa type of vector most commonly used. However, the present invention alsoincludes other types of expression vectors such as viral vectorsproviding an equivalent function (for example, an adenoviral vector, anadeno-associated viral (AAV) vector, a herpes viral vector, a retroviralvector, a lentiviral vector, and a baculoviral vector). Preferably, alentiviral vector may be used. Transformation includes any method ofintroducing nucleic acids into organisms, cells, tissues or organs andmay be performed by selecting a suitable standard technique depending onthe type of host cell as known in the art. Examples of this methodinclude electroporation, protoplast fusion, calcium phosphate (CaPO₄)precipitation, calcium chloride (CaCl₂)) precipitation, agitation byusing silicon carbide fiber, agrobacterium-mediated transformation, andPEG, dextran sulfate, Lipofectamine, and the like, but are not limitedthereto.

According to an embodiment of the present invention, artificialantigen-presenting cells may be produced by preparing each cDNA throughPCR amplification for genes of human HLA-A2 and co-stimulatory moleculesCD80, CD83, and 4-1BBL, inserting each cDNA into each lentiviral vector,and co-transducing these into K562 cells, and exosomes may be isolatedby subjecting the cells to ultracentrifugation.

The exosome of the present invention stably expresses HLA andco-stimulatory molecules CD32, CD80, CD83, and 4-1BBL and expressesCD63, CD81, ICAM, CD9, CD63, and CD82 as typical markers of exosomes,but expresses FLOT-1, ALIX, EpCAM, ANNXA5, and TSG101 at low levels.These markers are not present in the cell membrane of K562 cells whichare mother cells of the artificial antigen-presenting cells or areexpressed at low levels.

Further, the exosome of the present invention may additionally expressone or more selected from the group consisting of CD40L, CD70, and OX40Las a co-stimulatory molecule.

As used herein, “to sensitize an exosome with a substance” refers toreacting the exosome with the substance, and preferably refers todirectly or indirectly presenting the substance on the surface of theexosome. As used herein, the substance refers to an antigen, and the“foreign antigen” is an antigen which the cell itself does not possess,and cells may be sensitized by delivery of the antigen thereto orcontact with the antigen. As the delivery, it is possible to useelectroporation, transfection, and the like by pulse energy withoutlimitation. The contact may incubate an antigen and an exosome for acertain period of time.

For example, electroporation may use square wave pulses at a fieldstrength of 100 to 150 volts/mm gap width for 2 to 10 ms (for example,400 to 600 V for a gap of 4 mm), but may be appropriately adjusted atthe level of a person with ordinary skill in the art.

As used herein, the term “antigen” is well known in the art, andincludes not only all molecules capable of binding to antibodies, butalso epitopes, peptide fragments of antigens capable of binding to MEWmolecules, and immunogens. In the present invention, as the antigens,tumor antigens, pathogenic antigens, autoantibodies (normal ordiseased), or the like are used, but the antigens is not limitedthereto.

The tumor antigen refers to an antigen associated with tumors as a tumorassociated antigen (TAA). Examples of well-known TAAs include ovalbumin,survivin, gp75, gp1OO, MDM2, MART-1, MAGE-1, MAGE-3, tyrosinase,telomerase, her-2/neu, α-1 fetoprotein, G250, NY-ESO-1, and the like.Sequences of some peptides fragments of the TAA binding to MHC moleculesinclude Ova₂₅₇ (SIINFEKL: SEQ ID NO: 9), tyrosinase-related protein 1₄₅₅(Trp1₄₅₅; TAPDNLGYA: SEQ ID NO: 10), Trp2₁₈₀ (SVYDFFVWL: SEQ ID NO: 11),and gp100₂₅ (gp10025; EGSRNQDWL: SEQ ID NO: 12), a MAGE 1 nonapeptide(EADPTGHSY: SEQ ID NO: 13), a MART-APL peptide (LAGIGILTV: SEQ ID NO:14), a natural peptide (AAGIGILTV: SEQ ID NO: 15) or a PSA-1 peptide(FLTPKKLQCV: SEQ ID NO: 16), and the like. Additional sequences of thetumor associated peptides and antigens are known to those skilled in theart.

Examples of tumors associated with the tumor antigen include a solidtumor, a liquid tumor, hematologic tumor, renal cell cancer, melanoma,breast cancer, prostate cancer, testicular cancer, bladder cancer,ovarian cancer, cervical cancer, stomach cancer, esophageal cancer,pancreatic cancer, lung cancer, neuroblastoma, glioblastoma,retinoblastoma, leukemia, myeloma, lymphoma, hepatoma, adenocarcinoma,sarcoma, a malignant tumor (carcinoma), blastoma, and the like.

The pathogen antigen refers to any organism or virus causing a diseaseand also to attenuated derivatives thereof. The term “pathogen” refersto any virus or organism which is involved in the etiology of a diseaseand also to attenuated derivatives thereof. Such pathogens includebacteria, protozoan, fungal and viral pathogens, for example,Helicobacter sp., for example, Helicobacter pylori, Salmonella sp.,Shigella sp., Enterobacter sp., Campylobacter sp., various mycobacteria,for example, Mycobacterium leprae, Mycobacterium tuberculosis, Bacillusanthracis, Yersinia pestis, Francisella tularensis, Brucella sp.,Leptospira interrogans, Staphylococcus sp., for example, S. aureus,Streptococcus sp., Clostridum sp., Candida albicans, Plasmodium sp.,Leishmania sp., Trypanosoma sp., human immunodeficiency virus (HIV),hepatitis C virus (HCV), human papilloma virus (HPV), cytomegalovirus(CMV), HTLV, herpes virus (for example, herpes simplex virus type 1,herpes simplex virus type 2, coronavirus, varicella-zoster virus, andEpstein-Barr virus), papilloma virus, influenza virus, hepatitis Bvirus, poliomyelitis virus, measles virus; mumps virus, or rubellavirus, but are not limited thereto.

Examples of the autoantibody include an anti-nuclear antibody, ananti-γ-globulin antibody, an antibody against an autoblood component, oran antibody against an autoorgan, but are not particularly limitedthereto. When the autoantibody is used as a foreign antigen, the CD4 Tcell vaccine may induce strong anti-tumor immunity, and, thus, it can beeffective in overcoming potential immunological tolerance to aself-antigen expressed in normal tissue.

The exosomes sensitized with the antigen of the present invention arecharacterized in that the exosomes induce proliferation ofantigen-specific CD8+ T cells and directly stimulate CD8+ T cells, orwhen K562 cells and the exosome are washed after being co-cultured, andthen treated with the antigen, co-stimulatory molecules and HLAexpressed from the exosome are delivered to K562 cells, and the K562cells may stimulate CD8+ T cells, and thus may indirectly stimulate CD8+T cells by delivering a surface substance to other cells.

The stimulation of CD8+ T cells by the exosome is similar to the levelof that of dendritic cells.

Since the exosome of the present invention is sensitized with a foreignantigen while overexpressing co-stimulatory molecules to improve anantigen-specific T cell response, the exosome is effective in treatingtumors, pathogen infections, or autoimmune diseases according to thetype of foreign antigen.

Accordingly, the present invention provides an immunotherapeutic agentincluding the exosome.

The immunotherapeutic agent according to the present invention mayincrease an immune response or selectively elevate a portion of theimmune response preferred for the treatment or prevention of a specificdisease, infection or disorder.

Based on this, the present invention provides a vaccine for preventingtumors, pathogen infections, or autoimmune diseases, or a pharmaceuticalcomposition for treating tumors, pathogen infections, or autoimmunediseases, including the exosome.

For example, examples of the tumor include a renal cell tumor, melanoma,chronic lymphocytic leukemia, breast cancer, lung cancer, prostatecancer, ovarian cancer, colorectal cancer, or the like, but are notparticularly limited thereto.

Preferred examples of the pathogen infection include HIV, HCV, and thelike, but are not particularly limited.

Preferred examples of the autoimmune disease include systemic lupuserythmatosus (SLE), rheumatoid arthritis (RA), rheumatoid fever, and thelike, but are not particularly limited thereto.

The vaccine of the present invention may include all immunizationmethods performed by single administration and immunization methodsperformed by continuous administration.

The pharmaceutical composition may include an active ingredient with anactive or inert pharmaceutically acceptable carrier, making thecomposition suitable for diagnostic or therapeutic use in vitro, in vivoor ex vivo.

The pharmaceutically acceptable carrier includes any pharmaceuticalcarrier compatible with T cells, such as a phosphate buffered salinesolution and a protein excipient including serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. For examples of carriers, stabilizers and adjuvants, referto Martin REMINGTON'S PHARM. SCI, 18th Ed. (Mack Publ. Co., Easton(1995)) and the “PHYSICIAN'S DESK REFERENCE”, 58nd Ed., MedicalEconomics, Montvale, N.J. (2004). The term “carrier” may include abuffer or a pH adjusting agent, and typically, the buffer is a saltprepared from an organic acid or base. A representative buffer includesorganic acid salts such as salts of citric acid, salts of ascorbic acid,salts of gluconic acid, salts of carbonic acid, salts of tartaric acid,salts of succinic acid, salts of acetic acid, or salts of phthalic acid;Tris, tromethamine hydrochloride, or phosphate buffers. Additionalcarriers include a polymeric excipient/additive such aspolyvinylpyrrolidone, Ficoll (a polymeric sugar), a dextrate (forexample, cyclodextrin, for example,2-hydroxypropyl-quadrature,-cyclodextrin), polyethylene glycol, anantioxidant, an antistatic agent, a surfactant (for example, apolysorbate such as “TWEEN 20” and “TWEEN 80”), a lipid (for example,phospholipid, fatty acid), a steroid (for example, cholesterol), and achelating agent (for example, EDTA). Agents for preventing or reducingice formation may be included.

The pharmaceutical composition of the present invention may be preparedin various formulations as appropriate. For example, a formulationsuitable for parenteral administration, such as by intratumoral,intraarterial (in the joints), intravenous, intramuscular, intradermal,intraperitoneal, intranodal and subcutaneous routes, and carriersinclude an antioxidant, a buffer, a bacteriostat, and a solute thatrenders the formulation isotonic with the blood of an intendedrecipient, and an aqueous and non-aqueous sterile suspension that mayinclude a suspending agent, a solubilizer, a thickening agent, astabilizer, and a preservative. Intravenous or intraperitonealadministration is a preferred method. The dose of cells administered toa subject is in an amount, effective to achieve a desired beneficialtherapeutic response in the subject over time, or to inhibit growth ofcancer cells, or to inhibit infection. For example, the administrationmay be performed by a method of obtaining and storing a blood samplefrom a subject prior to infusion and by using the blood samples forsubsequent analysis and comparison. In general, at least about 1×10⁴ to1×10⁶ and typically, 1×10⁸ to 1×10¹⁰ cells may be infused intravenouslyor intraperitoneally into a 70 kg patient over roughly 60 to 120minutes. For administration, cells of the present invention areadministered at a rate determined by the LD-50 (or other methods ofmeasuring toxicity) according to the cell type and the side-effectsaccording to the cell type at various concentrations, in considerationof the overall health status and body weight of the subject.Administration may be accomplished via single or divided doses. Theexosome of the present invention may supplement other treatments for aspecific condition by using a known conventional therapeutic methodincluding a cytotoxic agent, a nucleotide analog and a biologic responsemodifier. Similarly, the biological response modifier may be optionallyadded to treatment by the exosome of the present invention.

Further, the present invention provides a method for proliferating Tcells, the method including a step of co-culturing the exosome and anyone T cell of a CD4 T cell, a CD8 T cell, or a γδT cell.

The present invention also provides a method for preparing cytotoxic Tcells in vitro, the method including a step of stimulating any one of aCD4 T cell, a CD8 T cell, or a γδT cell with the exosome sensitized withone or more antigens selected from the group consisting of a tumorantigen, a pathogen antigen, and an autoantibody.

The exosome of the present invention may proliferate the T cells whenco-cultured with the CD4 T cell, the CD8 T cell, or the γδT cell. Inaddition, when the T cells are stimulated by an exosome sensitized withan antigen, antigen-specific cytotoxic T cells may be produced.

The stimulation or co-culturing of the CD4 T cell, the CD8 T cell, orthe γδT cell by or with the exosome is performed in a cell culturemedium supplemented with Interleukin-2 (IL-2) in the absence of animmunostimulatory ligand.

The cell culture medium may be a safe medium for animal cell culture.Examples of the safe medium include Dulbecco's Modified Eagle's Medium(DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME),RPMI1640, F-10, F-12, α-Minimal Essential Medium (α-MEM), Glasgow'sMinimal Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium,and the like, but are not limited thereto.

The IL-2 may be added at a concentration of 20 to 100 IU/mL.

The stimulation by using the exosome may be performed for 4 days to 10days, but is not particularly limited thereto.

The culture conditions may be performed at a flow rate amount of 5 to15% carbon dioxide and at 35 to 37° C. in a CO₂ incubator, but are notparticularly limited thereto.

Hereinafter, the present invention will be described in more detailthrough the Examples according to the present invention, but the scopeof the present invention is not limited by the Examples suggested below.

EXAMPLES <Example 1> Preparation of Exosome

(Cells)

The use of human materials was reviewed and approved by theInstitutional Review Board of College of Medicine of the CatholicUniversity of Korea. PBLs were collected from healthy volunteers byusing Ficoll-Hypaque (GE Healthcare, Pittsburgh, Pa., USA). K562 celllines were obtained from the American Type Culture Collection (Manassas,Va., USA). All of the cell lines were cultured as recommended by theprovider.

(Establishment of Artificial APCs)

HLA-A2, CD80, 4-1BBL, and CD83 cDNA were individually cloned into apcDNA 3.1 vector (Invitrogen, Carlsbad, Calif., USA) and sequentiallytransfected into K562 cells by using the Nucleofector Kit (Lonza, Basel,Switzerland), according to the manufacturer's instructions.

K 562 cells were transfected with pcDNA3-CD32, capable of being loadedwith anti-CD3 antibodies to produce KEX. Moreover, K562 cells weretransfected with 4-1BBL, CD80, and CD83 to produce CoEX, respectively.Here, CoEX-A2 was further produced by further transfection of HLA-A2.Stable transfectants were selected by using 1 mg/ml G418 and FACS cellsorting by each antibody staining.

(Isolation and Culturing of CD8+ T Cells)

Human peripheral blood was obtained from 5 healthy HLA-A2 volunteers andmononuclear cells were isolated by using Ficoll-Hypaque (AmershamPharmacia Biotech, Piscataway, N.J., USA) density gradientcentrifugation. The HLA-A subtypes of the volunteers were determined bysequence-based typing in an HLA laboratory. Consent forms and approvalfor this study were obtained from the volunteers and the InstitutionalReview Board of College of Medicine of the Catholic University of Korea.Following density separation, CD8+ T cells (purity: up to 95%) wereisolated by using MACS isolation kits (Miltenyi Biotec, BergischGladbach, Germany).

(Culturing and Antigen Pulsing of Dendritic Cells)

Immature DCs were produced from CD14+ monocytes by culturing in a RPMI1640 medium supplemented with 10% fetal bovine serum, 100 ng/mL ofGM-CSF (GM-CSF; Endogen, Woburn, Mass., USA), and 50 ng/mL of IL-4(IL-4; Genzyme, Cambridge, Mass., USA) in a humidified incubator at 37°C. with 5% CO₂ while exchanging the media every 3 days for 6 to 7 days.Immature DCs were harvested and then infected with Adv-survivin with aMOI of 500 and/or a recombinant pp65 protein of 2 L/10⁶ DCs (MiltenyiBiotec, Bergisch Gladbach, Germany) at 37° C. for 3 hours. After antigenloading, DCs were matured for 24 hours by 100 ng/mL of TNF-α and 100ng/mL of LPS.

(Exosome Purification and Antibody Array)

The procedure for exosome isolation was based on a previously disclosedexosome purification method using ultracentrifugation (Current Protocolsin Cell Biology (2006) 3.22.1-3.22.29). When cells reached 90%confluency, the media were exchanged, and cultured after 72 hours, andthen the supernatants were collected. The collected supernatants werecentrifuged at 500 g for 10 minutes to remove cell debris. The clarifiedsupernatant was concentrated to a volume of 500 mL by centrifugation for10 minutes, filtered through a 0.22 mm filter and then centrifuged at100,000 g for 1 hour and 30 minutes. The exosomal pellet at the bottomof centrifugal tube was washed and eluted with PBS. Finally, the contentof exosomal protein was determined by using the bicinchoninic acid (BCA)assay and NanoDrop ND-1000 (NanoDrop Technologies, Montchanin, Del.,USA).

Known exosomal markers were detected by using the Exo-Check antibodyarray (System Biosciences, Mountain View, Calif.). Briefly, exosomalprotein lysates were prepared by adding 600 μL of an exosome lysisbuffer to 300 μg of exosomal protein. The antibody membrane array wasplaced in distilled water at room temperature for 2 minutes. Exosomallysate/binding mixture was added to the antibody membrane and thenincubated on a shaker at 41° C. overnight. After washing with an arraywash buffer, a detection buffer was added to the membrane and incubatedat room temperature for 2 hours. The final signal was analyzed bywashing the membrane twice with the wash buffer, developing themembrane, and then exposing the membrane.

(Flow Cytometry)

1) Surface Marker Analysis

Exosomes were coupled to the following modified beads as previouslydisclosed (Thery, C et al. Journal of immunology, vol. 166, pp.7309-′7318, 2001). Purified exosomes (30 μg) were incubated with 4-mmdiameter aldehyde/sulfate latex beads (Interfacial Dynamics, Portland,Oreg.) for 15 minutes at room temperature. Subsequently, the exosomeswere diluted with PBS, and the coupling reaction was continued for anadditional 2 hours. The reaction was stopped by adding 100 mM glycinethereto. Coated beads were washed three times in PBS and stained withspecific antibodies. The following fluorescence-conjugated antibodieswere used to examine the surface expression of co-stimulatory moleculesor exosomal markers: aCD9(SN4 C3 3A2), aCD63(H5C6), aCD82(ASL-24),a4-1BBL(5F4), aCD83(HB15e), aCD80(2D10), and HLA-A2(BB7.2). Allantibodies were purchased from BioLegend (San Diego, Calif., USA). Inbrief, 1×10⁶ viable T cells were washed twice with PBS containing 2%fetal bovine serum and incubated with 0.5 mg of the associatedantibodies for 20 minutes.

2) CD8+ T Cell Proliferation Assays

CFSE-labeled CD8+ T cells were stimulated by the presence of 50 μg/mL ofCoEX-A2, CoEX, KEX-A2, and KEX for 130 hours. After stimulation for 130hours, cells were harvested and stained with a PE-anti-human CD8antibody. The stained cells were applied to FACScanto II. Data wereanalyzed using ModFIT LT software (Verity Software House Inc., Topsham,Me.).

3) Cytotoxicity Testing

After antigen pulsing, DEX or CoEX-A2 was cultured with CD8+ T cells.CD8+ T cells purified to a final concentration of 2.0×10⁶/well in a24-well plate and DEX or CoEX-A2 were co-cultured in a RPMI 1640supplemented with 10% fetal bovine serum (Gibco-BRL), 2 mM L-glutamine,and 1% penicillin-streptomycin (Cambrex). On day 7, the cells wereharvested for re-stimulation. 20 U/mL IL-2 (Endogen) was added to thewells every 3 days, beginning on day 8. After 3 rounds of stimulation,the T cells were evaluated for antigen-specific immune responses.Expanded CD8+ T cells were evaluated by staining with 7-AAD after 6hours of post cultivation under the indicated experimental conditionsusing 100 μg CoEX-A2 or DEX in the presence of 20 IU/mL IL-2. Data wereanalyzed using FlowJo software (Tree Star, Otlen, Switzerland).

(ELISPOT Assay)

For ELISPOT assays, 5×10⁴ CD8+ T cells were added to each well ofMultiScreen hemagglutinin plates (Millipore, Bedford, Mass.) that werepre-coated with one among anti-IFN-γ (6 μg/mL) capture antibodies. After48 hours of incubation, culture plates were washed with an excess ofPBS_0.1% Tween 20, a detection antibody was added to the anti-IFN-γ (6μg/mL) capture antibody, and the plates were incubated at 4° C. for 24hours. After washing, streptavidin-horseradish peroxidase (1:1,000) wasadded thereto, and the plates were incubated at room temperature for 2hours. The plates were washed and developed using the peroxidasesubstrate 3-amino-9-ethylcarbazole (Sigma, Deisenhofen, Germany). Spotswere counted in an ELISPOT Reader (AID Elispot, Strassberg, Germany). Toestimate the number of antigen-responsive T cells, the number of spotsin wells containing no antigen (background) was subtracted from those ofthe experimental wells. For detection of cells secreting IFN-g, ELISPOTassays were performed using a BD Elispot assay kit (BD Biosciences), andthe procedure was performed according to the manufacturer'sinstructions. Peptides restricted to HLA-A2, p65₄₉₅₋₅₀₃ (NLVPMVATV: SEQID NO: 5), MP1₁₅₉₋₁₆₇ (YLQQNWWTL: SEQ ID NO: 6), MART1₂₆₋₃₅(ELAGI-GILTV: SEQ ID NO: 7), and WT₁₂₆₋₁₃₄ (RMFPNAPYL: SEQ ID NO: 8)were synthesized by AnyGen Co. Ltd. (Gwangju, South Korea). Briefly, Tcells were pulsed repeatedly three times with 10 g/mL of pp65, LMP1,MART1, and WT1 peptides at a ratio of 10:1, or incubated with unpulsedT2 cells. The number of spots corresponding to IFN-γ-secreting cells wascounted using AID-ELISPOT Reader.

(Statistical Analysis)

The results are expressed as mean±SEM. Statistical significance wasdetermined by the Student t test or by two-way ANOVA, followed by theTukey post hoc test to analyze clinical scores. Statistical significancewas accepted at p<0.05.

<Experimental Example 1> Characterization of Exosomes Derived fromGenetically Engineered K562 Cells

As illustrated in FIG. 1a , genetically engineered K562 cells stablyexpress various co-stimulatory molecules such as CD80, CD83, and CD137L(4-1BBL), CD32, and HLA-A2 genes. When genetically engineered K562 cellswere treated with pp65, CD8 T cells were stimulated, and then analysisby CFSE was performed, proliferation of viral antigen-specific cells canbe induced 5 days after culture (FIG. 1b ).

In order to isolate exosomes from a culture solution in which variousco-stimulatory molecules and HLA-A2 genes were expressed and confirm theexosomes, CD81, ICAM, CD63, ALIX, TSG101, EpCAM, and FLOT-1 as apositive control and CD9, CD63, and CD82 were analyzed by a Western blotmethod and a flow cytometry method, respectively. GM130 expressed inGolgi was used as a negative control.

As a result of the experiment, CoEX-A2 included HLA-A2, CD80, CD83, and4-1BBL at detectable levels (FIG. 1c ). Further, CoEX-A2 includedtypical exosomal marker proteins CD9, CD63, and CD82, but these markerswere not present in the cell membrane of K562 cells, or were expressedat low levels (FIG. 1d ). Finally, the exosome CoEX-A2 was positive forCD63, CD81, and ICAM, but FLOT-1, ALIX, EpCAM, ANNXA5, and TSG101 wereexpressed at low levels (FIG. 1e ).

<Experimental Example 2> Immunostimulatory Response Confirmation ofGenetically Engineered Exosomes

K 562 cells were transfected with pcDNA3-CD32, capable of being loadedwith anti-CD3 antibodies to produce CoEX. The first visible event in Tcell activation is the formation of clusters between CoEX and CD8+ Tcells. To assay for cluster formation, CD8+ T cells were cultured in thepresence or absence of soluble anti-CD3 antibodies, IL-2, and CoEX.Mixtures of CoEX and CD8+ T cells remained free of clusters in theabsence of anti-CD3 antibodies, but large clusters in mixtures of CoEXand CD8+ T cells were observed in the presence of anti-CD3 antibodies(not shown). Further, these clusters grew in size for 8 hours to 12hours. This effect of CoEX on the proliferation of cultured CD8+ T cellswas enhanced by incubation with OKT3 and IL-2. In addition, optimalproliferation of these cells was observed in the presence of OKT3, IL-2,and CoEX, except for KEX (FIG. 2a ). Furthermore, to confirm the degreeof effect of CoEX, the effect was compared with that of DYNABEADincluding CD3 and a co-stimulatory molecule CD28, and it was observedthat CFSE-labeled CD8+ T cells were activated at similar levels in bothgroups, and the proliferation degree of cells was also significantlyincreased (FIG. 2 b).

According to the stimulation procedural view disclosed in the left sideview of FIG. 3, after the number of γδT cells was increased by treatinghuman peripheral blood with zoledronic acid and IL-2 (1000 IU/mL) for 7days, γδT cells were stimulated for 14 days by using CoEX or feedercells CoAPC and OKT3 (0.5 μg/mL) from Day 7.

As illustrated in the right side view of FIG. 3, CoEX may proliferateγδT cells at the one-third fold level of CoAPC.

As illustrated in the bottom side view of FIG. 3, it was confirmed thatwhen CoEX was used, the purity of Vγ9+/Vδ2+ T cells was increased withthe passage of culture time.

CoEX-A2 stimulated pp65-specific CD8+ T cells at a level similar to thatof DEX. In contrast, an exosome (KEX) isolated from K562 cells, anexosome (CoEX) expressing only co-stimulatory molecules, and an exosome(KEX-A2) expressing only HLA-A2 did not exhibit a significant level ofimmunostimulatory response (FIG. 4a ).

Similarly, CoEX-A2 exhibited significant stimulation of CD8+ T cells ata level similar to that of DEX in a proliferation analysis using theCFSE dilution method, unlike the other exosome groups (FIG. 4b ).

To find a suitable concentration of CoEX-A2 for stimulation of viralpeptide-specific CD8+ T cells, CoEX-A2 was cultured with CD8+ T cells inthe presence of a pp65 peptide.

As illustrated in FIG. 5, CoEX-A2 induced similar levels of CD8+ T cellstimulation at 50 μg/mL and 5 μg/mL, but at 0.5 μg/mL, the frequency ofIFN-γ-secreting T cells was even lower. Further, treatment with CoEX-A2exhibited an even lower level of stimulatory activity than engineeredK562 cells. From these results, 5 μg/mL was selected as a concentrationof exosomes for the subsequent experiments.

T cell stimulation responses were confirmed by treating CD8+ T cellsobtained from 5 volunteers expressing HLA-A2 and two volunteersexpressing no HLA-A2 with CoEX-A2.

As illustrated in FIGS. 6A and 6B, as a result of stimulation withpp65₄₉₅₋₅₀₃ (NLVPMVATV: SEQ ID NO: 5) and MART1₂₆₋₃₅ (ELAGIGILTV: SEQ IDNO: 7) peptides restricted to HLA-A2, cells harvested from healthyvolunteers expressing HLA-A2 produced pp65-specific CD8+ T cells andMART-1-specific CD8+ T cells at the number of 100 or more and less than100 per 10⁵ cells, respectively. Meanwhile, there was no response in thehealthy volunteer sample expressing no HLA-A2. Therefore, theproliferation of antigen-specific CD8+ T cells was confirmed using bothviral antigens and tumor antigens, as restricted to HLA-A2.

<Experimental Example 3> Direct and Indirect Antigen Presentation andCo-Stimulatory Transfer of CoEX-A2

In order to confirm direct and indirect stimulation responses of CoEX-A2to pp65-specific CD8+ T cells, experiments were carried out by settingGroup 1 (CD8, pp65, and CoEX-A2 mixed at one time), Group 2 (pp65 wasput in CD8 and washed, followed by treatment with CoEX-A2), Group 3(CoEX-A2 treated with pp65, washed and CD8+ T cells treated), and Group4 (CD8 treated with only pp65).

As illustrated in FIG. 7a , when the four groups were treated with theexosome and the stimulation of IFN-γ-secreting T cells was confirmed,Group 1 exhibited the strongest response because the exosome transferredco-stimulatory molecules and HLA-A2 to CD8+ T cells and also directlystimulated CD8+ T cells, and Group 2 exhibited an intermediate level ofimmune response because CoEX-A2 was transferred to CD8+ T cells. InGroup 3, a direct immune response of CoEX-A2 was confirmed.

In order to confirm the migration of HLA-A2 and co-stimulatory moleculesexpressed in the exosome, K562 cells and the exosome were co-culturedand washed, and then treated with antigens. Through ELISPOT, thisexperiment confirmed that the co-stimulatory molecules and HLA expressedin the exosome were transferred to K562, and these K562 cells couldstimulate CD8+ T cells (FIG. 7b ). As a result, it can be seen thatCoEX-A2 may directly stimulate CD8+ T cells or stimulate CD8+ T cells bydelivering surface substances to other cells.

<Experimental Example 4> Comparison of CoEX-A2 and DEX in CD8+ T CellStimulation

Finally, the inductions of viral and tumor antigen-specific CD8+ T cellsof CoEX-A2 and DEX were compared.

As illustrated in FIGS. 8a to 8c , the proliferation level of CD8+ Tcells was induced to a level similar to that of DEX when CD8+ T cellswere treated with CoEX-A2. Even when cultured for 14 days or more, pp65-and MART1-specific CD8+ T cells were increased to an extent similar toDEX when treated with CoEX-A2.

In order to confirm the cytotoxicity of CD8+ T cells induced byCoEX-A2/pp65 and DEX/pp65, T cells treated with a pp65 peptide weretested as a target. The death rate of target cells was confirmed bystaining with 7-AAD (viability dye) (FIG. 9a ).

Further, CoEX-A2/pp65 CD8+ T cells and DEX/pp65 CD8+ T cells were testedby setting the ratio of effector:target cells at 6.25:1, 12.5:1, 25:1,50:1, 100:1, and 200:1.

As a result of the experiment, there was no significant differencebetween CD8+ T cell groups according to the ratio of effector:targetcells (FIG. 9b ).

From the result, CoEX-A2 could produce antigen-specific CD8+ T cells invitro, and direct and indirect stimulation abilities of these exosomeswere similar to that of DEX.

The present invention may be applied to the field of prevention ortreatment of tumors, pathogen infections, or autoimmune diseases.

1. An exosome which expresses a human leukocyte antigen (HLA), CD32,CD80, CD83, and 4-1BBL.
 2. The exosome of claim 1, wherein the exosomeis derived from artificial antigen-presenting cells prepared byintroducing nucleic acids encoding HLA, CD32, CD80, CD83, and 4-1BBLinto cells which do not express HLA Class I and II molecules.
 3. Theexosome of claim 2, wherein the cells which do not express HLA Class Iand II molecules are any one of K562 cells and modified 293T cells. 4.The exosome of claim 2, wherein the nucleic acid is inserted into aviral vector and delivered into cells.
 5. The exosome of claim 2,wherein the nucleic acid encoding HLA is as set forth in SEQ ID NO: 1.6. The exosome of claim 2, wherein the nucleic acid encoding CD80 is asset forth in SEQ ID NO:
 2. 7. The exosome of claim 2, wherein thenucleic acid encoding CD83 is as set forth in SEQ ID NO:
 3. 8. Theexosome of claim 2, wherein the nucleic acid encoding 4-1BBL is as setforth in SEQ ID NO:
 4. 9. The exosome of claim 1, wherein the exosomefurther expresses one or more selected from the group consisting ofCD40L, CD70, and OX40L.
 10. The exosome of claim 1, wherein the exosomeis sensitized with one or more antigens selected from the groupconsisting of a tumor antigen, a pathogen antigen, and an autoantibody.11-13. (canceled)
 14. A method for proliferating T cells comprising astep of co-culturing the exosome according to claim 1 and any one T cellof a CD4 T cell, a CD8 T cell, or a γδT cell.
 15. A method for preparingcytotoxic T cells in vitro comprising a step of stimulating any one of aCD4 T cell, a CD8 T cell, or a γδT cell with the exosome according toclaim 1 sensitized with one or more antigens selected from the groupconsisting of a tumor antigen, a pathogen antigen, and an autoantibody.